BR112019009430A2 - generation of human oocytes - Google Patents

Abstract

The present invention relates to methods of generating functional human oocytes after nuclear transfer of first polar body (pb1) genomes from metaphase II (mii) oocytes to enucleated donor mii cytoplasm (pbnt) and using mitochondrial replacement techniques to circumvent the transmission of mtdna disease from mother to child.

Description

"GENERATION OF HUMAN OOCYTES"

TECHNICAL FIELD [001] Generally, the field involves the generation of human oocytes in vitro through the transfer of nuclear material. More specifically, the field involves the generation of human oocytes through nuclear transfer of polar bodies to enucleated donor oocytes and / or the transfer of nuclear material from an oocyte containing mitochondrial DNA mutations to an oocyte without these mutations.

BACKGROUND OF THE INVENTION [002] Infertility attributed to male and female factors affects millions of families worldwide and while assisted reproductive technologies (ARTs) can work around many cases (Huang JY and Rosenwaks Z, Meth Mol Biol 1154, 171 a 231 (2014); incorporated by reference here; Leridon H, Human Reproduction 19, 1548 to 1553 (2004); Trounson A and Mohr L, Nature 305, 707 to 709 (1983); all of which are incorporated by reference), its effectiveness is particularly limited by the number and quality of oocytes that decrease with advanced maternal age (Leridon 2004 above). Therefore, the development of additional sources of competent oocytes, genetically related to patients, is desirable.

[003] Perhaps the final approach involves differentiating pluripotent stem cells into primordial germ cells (PGCs) and finally mature oocytes (Daley GQ et al., Science 316, 409 to 410 (2007); Handel MA et al., Cell 157, 1257 to 1261 (2014), Hayashi Y et al., Fertil Steril 97, 1250 to 1259 (2012), Matthews TJ et al., NCHS Data Brief 1-8 (2009), all of which are incorporated by reference) . However, the complexity of establishing an in vitro system to produce functional haploid human oocytes currently limits this application (Irie N et al., Cell 160, 253 to 268 (2015) and Sasaki K et al., Cell Stem Cell 17, 178 to 194 (2015), both of which are incorporated by reference).

[004] Maternally inherited mtDNA mutations can cause disease

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2/55 debilitating fatal or severe in children (Archer SL, Engl J Med 369, 2236 to 2251 (2013); Koopman WJ et al., N Engl J Med 366, 1132 to 1141 (2012); and Schon E et al., Nat Rev Genet 13, 878 to 890 (2012), all of which are incorporated by reference). The severity of the disease depends on the specific genetic mutation and the naturally occurring pathogenic mtDNA ratio (level of heteroplasm) in each cell and tissue (Wallace DC & Chalkia D, Cold Spr Harb Perspect Biol 5, a021220 (2013); incorporated here as reference). Pathogenic mtDNA mutations are relatively common with an estimated 778 affected children born each year in the United States (Gorman GS et al., N Engl J Med 372, 885 to 887 (2015); incorporated here by reference). Currently, there is no effective cure for patients with mtDNA mutations. Therefore, mitochondrial replacement techniques (MRTs) have been developed to prevent transmission of mutant mtDNA from mothers to their children (Wolf DP et al., Mol Med Trends 21.68 to 76 (2015); Wang T et al., Cell 157, 1591 1604 (2014), Tachibana M et al., Nature 493, 627 to 631 (2013), Craven L et al., Nature 465, 82 to 85 (2010) and Tachibana M et al., Nature 461,367 to 372 (2009) all which are hereby incorporated by reference). Regulatory agencies in the US and the UK are currently evaluating clinical MRT applications designed to assess efficacy and safety. To date, the clinical aspects of MRT have not been studied, including issues of patient eligibility, embryo development and maintenance of the post-MRT donor's mtDNA.

[005] There remains a need for improved methods of assisting with fertilization, implantation and successful pregnancy in patients with declining or otherwise incompetent oocytes.

SUMMARY OF THE INVENTION [006] Methods are provided here for preparing oocytes for fertilization and uterine implantation.

[007] Here again described the reconstruction of human haploid oocytes

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3/55 through the recycling of PB1 genomes through nuclear transfer in donor cytoplasm (Gurdon JB, Rambam Maimonides Med J 6 (2015), incorporated herein by reference).

[008] Oocyte defects are at the heart of some forms of infertility, and could potentially be treated therapeutically by alternative routes for the formation of oocytes. The generation of functional human oocytes after nuclear transfer of genomes from the first polar body (PB1) from metaphase II (Mil) oocytes to the enucleated donor Mil cytoplasm (polar body transfer, PBNT) is described here. The reconstructed oocytes supported the formation of meiotic spindles again and, after fertilization with sperm, the completion of meiosis and the formation of normal diploid zygotes. While PBNT zygotes evolved to blastocysts less frequently (42%) than controls (75%), genetic, epigenetic and transcriptional analyzes of PBNT and control embryonic stem cells (ESCs) indicated comparable numbers of structural variations and transcriptome profiles and similar DNA methylation. PB1 genetic material through introduction into the donor cytoplasm can provide oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

[009] This method allows the generation of additional oocytes corresponding to the patient for the treatment of infertility. Using each patient's Mil oocyte and its PB1 (plus donor cytoplasm), the patient-related oocytes / embryos can be theoretically duplicated. Realistically, the development of PBNT embryos for blastocysts is less. Thus, it can be estimated that the number of viable embryos for transfer and therefore pregnancy rates can increase by 40%.

[010] A method for generating a viable human oocyte is described here. The method involves enuclearing a first oocyte in the Mil stage, thus creating a cytoplasm, isolating a polar body from a second oocyte by aspiration, and placing

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4/55 the polar body in the perivitelline space of the cytoplasm. The method may also involve fertilizing the viable human oocyte, thereby creating an embryo. The method may also comprise implanting the embryo in a receptive (endometrial) uterine lining. Enucleation of the first oocyte can be performed in a medium comprising at least 10% human tubal fluid, a buffer, and cytochalasin B using a Polarizing Microscope Imaging System. The isolation of the polar body can be carried out in a medium comprising at least 10% of human tubal fluid, buffer, cytochalasin B, and the fusion with the donor cytoplasm can be induced with the HVJ-E extract (hemagglutinating virus from the envelope of Japan / Sendai virus).

[011] A method is provided to prepare an oocyte for fertilization, the method comprising the steps of:

a) isolate a first oocyte containing a polar body;

b) gain access through the pellucid zone of the first oocyte;

c) removing the polar body from the first oocyte;

d) removing the nuclear spindle from a second oocyte to create an enucleated cytoplasm;

e) placing the polar body removed from the first oocyte in the perivitelline space of the enucleated cytoplasm.

[012] A method for producing a human oocyte in vitro is also provided, the method comprising:

a) determine a box II mitochondrial DNA sequence of conserved sequence from a donor oocyte;

b) enuclear the donor's oocyte while the donor's oocyte is trapped in metaphase II, thus isolating the donor's nuclear genetic material;

c) introducing the donor's nuclear genetic material into an enucleated recipient's oocyte, provided that the donor's oocyte's mitochondrial DNA has the same

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5/55 box II genotype of conserved sequence than the oocyte of the recipient.

[013] Additional modalities of both methods above involve an additional step of fertilizing the newly created oocyte through in vitro fertilization. In additional modalities, the fertilized oocyte / embryo is cultured in the blastocyst stage before implantation in a receptive uterine / endometrial lining.

BRIEF DESCRIPTION OF THE DRAWINGS [014] Colored figures were sent as part of the original description and references to colors in the figures are provided in this description. Applicants reserve the right to submit color versions of the figures described in this document in later procedures.

[015] Figure 1A shows the generation of human haploid oocytes by PBNT. Ο PB1 of a donor oocyte is recovered and introduced into a Mil cytoplasm of the recipient. After fertilization, the zygotes of the PBNT are grown in the blastocyst stage before being transferred to a patient. Alternatively, they can be used to derive embryonic stem cells for further analysis.

[016] Figure 1B shows confocal microscopy detecting chromosomal structures of the meiotic spindle in PB1 (first panel from the left), Mil intact oocytes (second panel) and PBNT oocytes (third panel) marked with DAPI (blue) for DNA and a- and β-tubulins (green) for microtubules. It is observed the absence of meiotic spindle in PB1 (first panel) and abnormal spindles in prophase (third panel) or anaphase (last panel) in some PBNT oocytes. 5 pm scale bars.

[017] Figure 1C shows that most PB1s in recently recovered 1,000 human oocytes were intact with circular morphology (left panel). Other Mil oocytes contained fragmented or disintegrated PB1, unsuitable for PBNT (middle and right panels). Scale bar, 100 pm.

[018] Figure 1D shows the spindle formation (arrow), detected using the OosightTM imaging system, 60 min after ο PB1 was introduced into a

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6/55 enucleated Mil cytoplasm. Scale bar, 100 pm.

[019] Figure 1E shows the completion of meiosis and pronucleus formation in human PBNT oocytes after fertilization. The normal zygote with 2 pronuclei (2PN) and a second polar body (PB2) is observed in the left panel and 1PN, 3PN or 2PN abnormal without extrusion of PB2 in other images. Scale bars, 100 pm.

[020] Figure 1F shows an outbreak blastocyst from a PBNT zygote, with prominent ICM indicated by an asterisk. Scale bar, 100 pm.

[021] Figure 1G shows a summary of the in vitro fertilization and development of PBNT oocyte blastocysts. Error bars are mean ± SEM. Significance established with the Student t test. Control = Thousand oocytes intact.

[022] Figure 2A shows a cytogenetic G-band analysis of the PBNT 1, PBNT2, and hESO-14, hESO-15, hESO-16 and hESO-17 strains. Note that normal female karyotypes in PBNT1 and hESO-14 control and normal male karyotypes in hESO-16 control and hESO-17. PBNT2 exhibited an abnormal triploid karyotype while control hESO-15 had an abnormal karyotype with chromosome 21 monosomy.

[023] Figure 2B shows a Venn diagram showing the results of the structural variation analysis in PBNT 1, hESO-14 compared to the egg donor. PBNT1 (blue) and hESO-14 (red) showed similar numbers of SVs when compared to the egg donor.

[024] Figure 2C shows a Venn diagram showing the results of the structural variation analysis in PBNT 1, hESO-14 compared to the sperm donor. PBNT1 (blue) and hESO-14 (red) showed similar numbers of SVs when compared to the sperm donor.

[025] Figure 2D shows a summary of SVs in PBNT1 and hESO-14.

[026] Figure 3A shows the percentage of CGs methylated in PBNT1 and hESO-14 of the total CG sites.

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7/55 [027] Figure 3B shows rates of non-CG methylation in PBNT1 and hESO-14 among all non-CG sites.

[028] Figure 3C shows that a total of 116 CG DMRs were identified between PBNT1 and hESO-14. Genomic characteristics of DMRs (overlap of at least 1 bp) - CGI, CG islands; DHS, hypersensitivity sites to DNase I, TSS, transcription initiation sites; TES, transcription end sites.

[029] Figure 3D shows a representative screenshot showing a hypomethylated DMR CG in PBNT1 compared to hESO-14.

[030] Figure 3E is a set of screen shots showing four non-CG DMRs identified in PBNT1 compared to hESO-14.

[031] Figure 4A is a spot plot that summarizes the differential expression (DE) analysis. The log-fold variation of each of the 14877 genes used in the analysis is plotted against the average abundance (log counts per million) for each gene. The red dots represent genes with significantly different expression (FDR <0.05). The blue lines indicate 2-fold increases.

[032] Figure 4B is a heat map showing the expression profile of the 186 DE genes. Two distinct groups were apparent with genes expressed highly in PBNT1 cells compared to hESO-14 cells and vice versa (n = 6).

[033] Figure 5A shows histological analyzes of teratoma tumors produced after PBNT1 injections in SCID mice. Scale bars, 100 pm. The arrows indicate tissues similar to the neural crest (ectoderm), tissues similar to cartilage (mesoderm) and mucosal epithelium of intestine-like tissues (endoderm).

[034] Figure 5B shows histological analyzes of teratoma tumors produced after injections of hESO-14 in SCID mice. Scale bars, 100 pm. Arrows indicate tissues similar to the neural crest (ectoderm), tissues similar to cartilage (mesoderm) and mucosal epithelium of intestine-like tissues

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8/55 (endoderm).

[035] Figure 6A is a genealogy of families 1 and 2 of Leigh's Syndrome.

[036] Figure 6B is a genealogy of the Leigh Syndrome family 3 G13513A.

[037] Figure 6C is a genealogy of family 5 A3243G of the MELAS syndrome of great genealogy (see also Figure 10B).

[038] For all Figures 6A, 6B and 6C, the underlined levels of heteroplasm were obtained from previous reports. In all families, the levels of heteroplasm for mtDNA mutations were diverse between tissues and individuals, and associated with clinical disease. Filled black, diagnosed as a mitochondrial disease. LS = Leigh's syndrome, y = years of age, square = male; circle = female, B = blood; SF = skin fibroblasts; U = urine.

[039] Figure 7A is a set of graphs showing individual oocytes that harbor different levels of heteroplasm. cED = carrier oocyte donor.

[040] Figure 7B is a set of two graphs showing that the average level of heteroplasm in oocytes is correlated with levels in existing children, rather than those in carriers of oocyte carriers. Heteroplasm levels in patients and children were measured in the blood. R = Pearson's r-values.

[041] Figure 7C is a graph showing comparable mtDNA variants between oocytes with healthy mtDNA and those carrying mtDNA mutations (p> 0.05).

[042] Figure 7D is a graph showing that the total number of mtDNA copies was comparable in oocytes with healthy mtDNA and those carrying pathogenic mtDNA (p> 0.05). Karyoplasts were isolated from oocytes carrying ST, except for cED2 oocytes, which exhibited premature luteinization. The oocytes of healthy egg donors were one thousand mature, n = the number of oocytes. LS:

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9/55 Leigh syndrome; MELAS: mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, t test, ns = not significant (p> 0.05).

[043] Figure 8A is a set of graphs showing the development of the ST embryo after several experimental manipulations. Similar developmental results were observed between the groups, except for the normal fertilization rate, which was significantly lower in the frozen ST groups (p <0.05). Combinations = mixture of different mtDNA haplotypes between the karyoplast and the cytoplasm; Control intact = not manipulated, only ICSI.

[044] Figure 8B is a set of graphs showing that ST embryos carrying different types of mutations showed a similar pattern of development.

[045] Figure 8C is a set of graphs showing the development of the ST embryo as a function of the donor's mtDNA matching distances. The numbers at the top of the bars are percent (%) of embryonic development. N = the number of embryos, ns = not significant (p> 0.05).

[046] Figure 8D is a set of graphs of NPCs derived from ST ESCs with differences of 6 and 49 SNP in oxygen consumption rates (OCR) displayed from donor mtDNA similar to unhandled controls. However, NPCs with 33 SN Ps showed significantly reduced OCR compared to controls or donor or maternal mtDNA (p <0.05). The seahorse test was repeated 3 different times with 6 to 18 repetitions each time. See also Figure 13A. NPCs = neural progenitor cells. SNPs = single nucleotide polymorphisms, t-test or Unidirectional ANOVA.

[047] Figure 9A is an image of a gel showing polymorphism in the CSBII region of the donor mtDNA that results in reduced efficiency in generating mitochondrial RNA polymerase primers. The 311315CCCCC polymorphism is a shortening of the first G elongation in the G-quadruplex region

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10/55 of mtDNA (stretching G5 vs. G6), responsible for the termination of transcription and generation of replication primer in human mitochondria.

[048] Figure 9B is a graph showing rates of heteroplasm for maternal mtDNA (haplotype H49) gradually increased during the extended in vitro culture of 3243ST-ES1 and reached homoplasm.

[049] Figure 9C is a graph showing that the levels of heteroplasm also increased during the single ESC colony subculture.

[050] Figure 9D is a graph showing maintenance of stable heteroplasm in the progeny of individually cultured cells.

[051] Figure 9E is a graph showing faster rates of cell growth and proliferation in clones containing greater maternal mtDNA heteroplasm.

[052] Figure 9F is a set of three graphs showing changes in maternal mtDNA heteroplasm in reverse 3243ST-ES1, ST-ES7 and NTES8 cell lines during differentiation in vitro and in vivo. Unidirectional ANOVA. Mean ± SD.

[053] Figure 10A is a table showing families with mitochondrial disease and women of reproductive age recruited for MRT. All LS families had a severely affected child. F, family; non-sin, not synonymous; n / a, not applicable.

[054] Figure 10B shows that Family 5 was selected from an extensive MELAS syndrome genealogy. The clinical phenotypes of MELAS A3243G syndrome varied even with similar levels of heteroplasm. Squares, males vs. Circles, females; B = blood and U = urine, n / t, not tested. *, the first MELAS patient clinically diagnosed. Heteroplasmia in the blood (upper) and urine (lower).

[055] Figure 11A is a graph showing that the age of the oocyte donors was similar between the carrier and healthy groups.

[056] Figure 11B is a graph showing that the ovarian reserve has been

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11/55 significantly lower in carrier donors compared to healthy oocyte donors.

[057] Figure 11C is a graph showing that the count of antral follicles (AFC) was significantly lower in donor carriers compared to donors of healthy oocytes.

[058] Figure 11D is a graph showing that the duration of COS was longer in patients.

[059] Figure 11E is a graph showing that the peak of estradiol (E2) on hCG day tended to decrease in carriers.

[060] Figure 11F is a graph showing that total oocyte yield was significantly lower in carriers than healthy oocyte donors.

[061] Figure 11G is a graph showing that the number of mature oocytes was significantly lower in carrier donors than in healthy oocyte donors.

[062] Figure 11H is a table of baseline characteristics and cycle results. Patients had a higher BMI and lower levels of AMH. The lowest peak levels of estradiol were measured in cED1 and cED2. Birth control method: combined oral contraceptive. Medroxyprogesterone.

[063] Figure 111 is a set of two pie charts showing the analysis of heteroplastic mtDNA variants detected in Mil oocytes. Again, it indicates unique mutations found in individual oocytes; hereditary means mutations shared with other oocytes, siblings or egg donors, n = number of mutations in individual oocytes.

[064] Figure 11J is a set of two graphs that show that AMH levels, a measure of ovarian reserve, were not correlated with mtDNA copy number. n = the number of oocytes. ns = not significant.

[065] Figure 12A is a table showing the results of the sequencing

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12/55 of integral mtDNA performed to identify haplotypes from all egg donors and ST was performed to combine several haplotypes. Cyto, cytoplasm; Karyo, carioplasto.

[066] Figure 12B is a set of images showing normal fertilization (2PN, left) and abnormal fertilization (1 or 3 PN). See the arrows.

[067] Figure 12C is a table showing that the ST carrier showed greater abnormal fertilization than the controls. A 3 PN zygote in the ST control group evolved into a blastocyst and exhibited a 69, XXY karyotype. * p <0.05.

[068] Figure 12D is a table showing that the rate of blastulation was similar between cytoplasm vitrified with fresh spindles and spindles vitrified with fresh cytoplasm (p> 0.05).

[069] Figure 12E is a set of graphs showing the development of the embryo with mitochondrial DNA from different donors.

[070] Figure 13A is a set of 15 graphs showing the results in which energy metabolism (OCR / ECAR) was measured and compared between MRT-derived neural progenitor cells (NPCs) and donor or mtDNA control embryos or maternal mtDNA. OCR = oxygen consumption rate (representing OXPHOS), ECAR = extracellular acidification rate (representing glycolysis). MRT NPCs ESCs with 6 and 49 SNP differences showed OCR comparable to NPCs with maternal mtDNA or donor mtDNA, except for maximum group breathing of 49 SNP. OCR has been reduced on NPCs with 33 SNPs. OCR data was normalized by the DNA content of living cells. * vs. host mtDNA, ** vs. donor mtDNA, *** vs. ST (p <0.05, t test or Unidirectional ANOVA, n = 16-18 technical replicates). Mean ± SEM.

[071] Figure 13B is a set of graphs showing the enzymatic activity of the mitochondrial respiratory chain in cells differentiated from ST-ESCs. Mitochondrial complex activities I and IV in differentiated cells of ST-ESCs

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13/55 donor mtDNA carriers with 49 differences in SNP were comparable to controls or donor mtDNA or maternal mtDNA.

[072] Figure 14A is a table showing a summary of the in vitro and in vivo differentiation of ESCs derived from ST or SCNT embryos carrying donor mtDNA. Similar to the control of IVF-ESCs, all MRT ESCs tested produced teratoma tumors in vivo and formed neural progenitor cells (NPCs) and cardiomyocytes in vitro.

[073] Figure 14B is a histological analysis of tissues differentiated from MRT-ESCs. Representative tissues were collected and used for mtDNA transport analysis and measurements of mitochondrial function.

[074] Figure 15A is a graph and table showing that the rate of aneuploidy in blastocysts, determined by the CGH arrangement, was not significantly different in the ST groups compared to the control.

[075] Figure 15B is a graph and table showing that a rate of Karyotype abnormality in ESCs determined by G-band analysis was also comparable between the ST groups. Bars = mean ± s.d. Number inside bars = the number of blastocysts or ESC lines. Simple χ2 tests.

[076] Figure 16A is a set of two graphs showing that the heteroplasm of maternal mtDNA (haplotype X2c) increased during prolonged in vitro culture and reached homoplasm in clustered cultures or in subcultures of individual colonies.

[077] Figure 16B is a set of two graphs that show that the enzymatic activities of the respiratory chain 1 (COM I) and complex 4 (COM IV) enzymes were measured in fibroblasts carrying several human mtDNA haplotypes used in this study . No significant differences were observed.

[078] Figure 17 is a table that summarizes the CGH arrangement in blastocysts

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14/55 intact and ST.

[079] Figure 18 is a table that summarizes the G bands of ESC lines derived from control and ST blastocysts.

DETAILED DESCRIPTION [080] Here is provided a method for generating a viable human oocyte, the method comprising the steps of:

a) enuclear a first oocyte in the Mil stage, thus creating a cytoplasm;

b) isolating a polar body from a second oocyte by aspiration; and

c) placing the polar body in the perivitelline space, thus merging the polar body with the cytoplasm and generating the viable human oocyte.

[081] In other modalities, the methods included here involving polar body transplantation further comprise a stage of fertilizing the viable human oocyte via in vitro fertilization, thus creating an embryo. In other modalities, the method also involves implanting the embryo in a receptive endometrial uterine lining.

[082] In other modalities of the methods, the enucleation of the first oocyte is carried out in a medium comprising at least 10% of human tubal fluid, HEPES buffer and cytochalasin B. In other modalities, the enucleation step is completed using an Imaging System Polarization Microscope.

[083] In yet other modalities, the method step of isolating the polar body is carried out in a medium comprising at least 10% of human tubal fluid, HEPES buffer, cytochalasin B and fusion with donor cytoplasm induced with HVJ-E extract .

[084] In other modalities, the pellucid zone is opened using methods selected from zone cutting, laser drilling (such as with a compact nitrogen laser) and acid drilling (such as with Tyrode's acidic medium).

[085] A method for producing a human oocyte in vitro is also provided,

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15/55 comprising:

a) determine a mitochondrial DNA sequence from the conserved sequence II box of a donor oocyte;

b) enuclear the donor's oocyte while the donor's oocyte is trapped in metaphase II, thus isolating the donor's nuclear genetic material;

c) introducing the donor's nuclear genetic material into an enucleated recipient's oocyte, provided that the donor's oocyte's mitochondrial DNA has the same conserved sequence II genotype as the recipient's oocyte.

[086] In some embodiments, the method immediately above is performed when the donor oocyte and the recipient oocyte both have the G5A8 genotype or both have the G6A8 genotype. In yet other modalities of the method, the mitochondrial DNA of the donor oocyte comprises a mutation that results in a mitochondrial disease, including but not limited to Leigh's Syndrome or MELAS.

[087] Also provided is a method of producing a human oocyte in vitro, the human oocyte produced comprising minimal maternal mitochondrial DNA and the method comprising the steps of:

a) determining the sequence of the main non-coding region of the mitochondrial DNA of a donor oocyte, wherein the main sequence of the non-coding region includes a D-loop region;

b) enuclear the donor's oocyte while the donor's oocyte is trapped in metaphase II, thus isolating the donor's nuclear genetic material; and

c) introduce the donor's nuclear genetic material into a recipient's oocyte, provided that the donor's oocyte's mitochondrial DNA has the same sequence as the main non-coding region of the recipient's oocyte.

[088] In additional modalities of the method immediately above, the donor oocyte and the recipient oocyte both have the mtDNA H56, H1b, U5a, F1a, X2c, D4a, H49 or B2k haplotype. In other modalities, mitochondrial DNA from the oocyte

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16/55 of the donor comprises a mutation that results in a mitochondrial disease, such as Leigh's syndrome or MELAS.

[089] Other mitochondrial diseases for which the use of the present methods can be considered include, but are not limited to, the possible occurrence of mitochondrial myopathy; diabetes mellitus and deafness; progressive childhood polyiodistrophy (Alpers disease); Leber's hereditary optic neuropathy; Barth Syndrome / Infantile Lethal Cardiomyopathy (SCI); neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); myioneurogenic gastrointestinal encephalopathy (MNGIE); carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme deficiency Q10; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency / COX deficiency; complex V deficiency; myoclonic epilepsy with irregular red fibers (MERRF); mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS); depletion of mtDNA; Chronic Progressive External Ophthalmoplegia Syndrome (CPEO); CPT I deficiency; CPT II deficiency; Kearns-Sayre syndrome (KSS); lactic acidosis; LBSL-Leukodystrophy; Long-chain Acyl-CoA dehydrogenase (LCAD) deficiency; LCHAD; Luft's disease; Multiple deficiency of Acyl-CoA Dehydrogenase; Short Chain Acyl-CoA Dehydrogenase Deficiency; Deficiency of Medium Chain Acyl-CoA Dehydrogenase; Acyl-CoA Deficiency Very Long Chain Dehydrogenase; Mitochondrial Encephalomyopathy Lactic Acidosis and Stroke-like Episodes (MELAS); Mitochondrial Recessive Ataxia Syndrome; Mitochondrial Cytopathy; Depletion of mitochondrial DNA; mitochondrial encephalopathy; Mioneurogastrointestinal Disorder and Encephalopathy; Pearson's syndrome; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; and POLG2 mutations.

Definitions [090] The term “polar body” refers to haploid cells, significantly

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17/55 smaller than an oocyte that is formed together with an oocyte during oogenesis. In particular, the polar bodies include half of the diploid chromosome set not included in the oocyte after meiotic division. Polar bodies comprise polynucleotides (for example, DNA) that encode information about the individual. Nuclear genetic material includes, but is not limited to, chromosomes and chromatin. The term includes nuclear genetic material produced by meiotic cell division, such as division or a diploid cell, to a haploid oocyte. Thus, a cell includes nuclear genetic material derived from a donor polar body if the polar body has been transferred to an enucleated cytoplasm via somatic cell nuclear transfer.

[091] The term "oocyte" refers to a female gamete or germ cell involved in reproduction, also called an egg. A mature oocyte is a haploid cell with a single set of maternal chromosomes (23, X in a human primate) and is disrupted at metaphase II. A "hybrid" oocyte has the cytoplasm of a first primate oocyte (called a "receptor"), but does not have the nuclear genetic material of the receptor; it has nuclear genetic material from another oocyte or a polar body and is called the "donor" oocyte.

[092] Meiosis is a process of reducing division in which the number of chromosomes per cell is reduced by half. In animals, meiosis always results in the formation of gametes. During meiosis, the genome of a diploid germ cell, which is composed of long segments of DNA packed into chromosomes, undergoes DNA replication followed by two cycles of division, resulting in four haploid cells. Each of these cells contains a complete set of chromosomes, or half the genetic content of the original cell. Meiosis I separates homologous chromosomes, producing two haploid cells (23 chromosomes, N in humans), so meiosis I is called a reducing division. A regular diploid human cell contains 46 chromosomes and is considered 2N

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18/55 because it contains 23 pairs of homologous chromosomes. However, after meiosis I, although the cell contains 46 chromosomes, it is considered only N, because later, in anaphase I, the sister chromatids will remain together as the spindle pulls the pair towards the pole of the new cell. In meiosis II, an equational division similar to mitosis occurs, whereby the sister chromatids are finally separated, creating a total of 4 haploid cells (23 chromosomes, N) per daughter cell from the first division.

[093] A "meiotic spindle" is a structure that separates chromosomes into daughter cells during meiotic cell division. It is part of the cytoskeleton in eukaryotic cells. The spindle apparatus includes the spindle microtubules, associated proteins, and any centrosomes or asters present in the spindle poles. The spindle apparatus is vaguely ellipsoid in shape and tapers at the ends, but spreads in the middle. In the broad medium portion, known as the median spindle, the antiparallel microtubules are grouped by kinesins. At the pointed ends, known as spindle poles, microtubules are nucleated by centrosomes in most animal cells.

[094] The term "mitochondrial DNA" or "mtDNA" refers to the DNA of the mitochondria, a structure located in the cell's cytoplasm and not in the nucleus (where all other chromosomes are located). In vivo, all mtDNA is inherited from the mother. There are 2 to 10 copies of the mtDNA genome in each mitochondria. Mitochondrial DNA is a circular double-stranded molecule. It is very small in relation to the chromosomes of the nucleus and includes only a limited number of genes, such as those that encode several subunits of the respiratory chain mitochondrial complex and the genes of some ribosomal RNAs and transfer RNAs. One cell includes mtDNA derived from mitochondria based on cytoplasmic continuation replication, which in the case of polar body transfer is based on the cytoplasm of the receptor.

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19/55 [095] The term “DNA methylation” refers to the post-synthetic addition of methyl groups to specific sites on DNA molecules; the reaction is catalyzed by enzymes called DNA methyltransferases that are specific for nucleotides and methylation position. In eukaryotes, methylation is involved in gene expression, and plays a role in a variety of epigenetic mechanisms, including X chromosome development, inactivation, genomic printing, DNA mutability, and uncontrolled cell growth in cancer.

[096] The term "X chromosome inactivation" refers to the inactivation of one of each pair of X chromosomes to form Barr's body in female mammalian somatic cells. Thus, tissues whose original zygote contained heterozygous genes that carry X should have individual cells expressing one or the other, but not both, of the gene products encoded by X. Inactivation is believed to occur early in development and lead to expression mosaicism of these genes in the body.

[097] The phrase “dosage compensation” refers to a mechanism that detects the dosage of genes and regulates expression accordingly. In mammals, there is monoallelic expression of X-linked genes that differ in dose between females (XX) and males (XY). "XIST" refers to a gene that encodes a large non-coding RNA that has been shown to be necessary for silencing the X-chromosome regulated by development in females. The XIST RNA is approximately 18 kb and is not translated, it is spliced, and polyadenylated. It is also organized in repetitive sequence blocks. In vivo, XIST RNA is found to be associated with the silenced X chromosome. XIST RNA expression is always limited by cis, and is associated with the silenced X chromosome in females.

[098] The term "effective amount" or "therapeutically effective amount" refers to the amount of agent or cell that is sufficient to prevent, treat,

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20/55 reduce and / or ameliorate the symptoms and / or underlying causes of any disorder or disease, or the amount of an agent sufficient to produce a desired effect in a cell. In one embodiment, a "therapeutically effective amount" is an amount of a cell or an agent sufficient to reduce or eliminate a symptom of a disease. In another embodiment, a therapeutically effective amount is an amount sufficient to overcome the disease itself.

[099] As used herein, the term "embryo" generally refers to a cell mass obtained by one or more divisions of a zygote or an activated oocyte with an artificially reprogrammed nucleus. A "morula" is the pre-implantation embryo 3-4 days after fertilization, when it is a solid mass, usually composed of 1232 cells (blastomeres). A "blastocyst" refers to a pre-implantation embryo in placental mammals (approximately 3 days after fertilization in mice, approximately 5 days after fertilization in humans) of approximately 30-150 cells. The blastocyst stage follows the morula stage, and can be distinguished by its unique morphology. The blastocyst is usually a sphere formed by a layer of cells (the trophectoderm), a fluid-filled cavity (the blastocele or blastocyst cavity), and a cluster of cells inside (the ICM).

[0100] "Nuclear transfer" refers to the insertion of a nucleus from the donor's polar body into a host cell of the enucleated recipient.

[0101] Unless otherwise explained, all technical and scientific terms used here have the same meaning as is commonly understood by one versed in the technique to which this description belongs. The singular terms “one”, “one” and “o”, ‘a” include plural referents, unless the context clearly indicates otherwise. Likewise, the word "or" must include "and", unless the context clearly indicates otherwise. It is further understood that all base sizes or sizes of amino acids, and all molecular weights or mass values

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21/55 molecular data for nucleic acids or polypeptides are approximate, and are provided for description. Quantities that are “approximately” a given range or numerical value include the exact range or numerical value. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this description, suitable methods and materials are described below. The term "comprises" means "includes". All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification, including explanations of terms, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0102] The development of normal and competent haploid oocytes depends on two consecutive divisions of meiotic cells in the absence of an intermediate DNA replication S phase (Petronczki et al., 2003). In mammals, primordial germ cells form primary oocytes that undergo chromosomal recombination during fetal development and then stop at the prophase of meiosis I. Meiosis is resumed, in turn, in selected primary oocytes after puberty, during periodic menstrual cycles, leading the completion of the first meiotic division, abstraction of PB1 and formation of a secondary oocyte that gets trapped again in the metaphase of meiosis II. A thousand resumes after ovulation, during fertilization, resulting in the segregation of the second polar body (PB2) and the formation of a haploid oocyte genome (Clift D and Schuh M, Mol Cell Biol 14, 549 to 562 (2013); incorporated here by reference). The contributions of haploid DNA from the sperm and egg mix during the preparation for the first mitotic division and completion of fertilization.

[0103] Interphasic nuclei of skin fibroblasts transplanted into oocytes Thousand enucleated humans (cytoplasts) undergo rapid remodeling and reprogramming of the cell cycle resulting in the formation of spindles attached to Mil oocyte type (Ma H and

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22/55 others, Nature 511, 177 to 183 (2014); Tachibana M et al., Cell 153, 1228 to 1238 (2013), which are hereby incorporated by reference). Residual meiotic activity in cytoplasts capable of forming Mil spindles again is crucial for reprogramming and subsequent embryonic development after nuclear somatic cell transfer (SCNT). As the main objective of human SCNT is to produce autologous ESCs, SCNT oocytes trapped in Mil are not fertilized with sperm, but are artificially activated to leave the meiotic prison, while the segregation of chromosomes during the second meiotic division is suppressed to preserve the diploid somatic cell. The potential for reprogramming the cytoplasm of the meiotic oocyte, as demonstrated by SCNT, suggested the possibility of producing haploid oocytes with donor genomes in the context of an infertility treatment. Previous studies in mice have shown that both PB1 and PB2 genomes can be reconstituted by transfer to compatible Mil or zygotic cytoplasm and contribute to the development of viable offspring (Wakayama T et al., J Reprod Fértil 110, 263 to 266 (1997); Wakayama T and Yanagimachi R, Biol of Reproduction 59, 100 to 104 (1998); incorporated herein by reference). However, these studies in mice have not been translated into other species, including primates and humans. The generation of human haploid oocytes is described here after the transfer of PB1 genomes to enucleated Mil oocytes and the induction of meiosis II again after sperm fertilization (Figure 1 A). The pre-implantation development of PBNT oocytes for blastocysts and ESCs has also been studied and their genetic and epigenetic properties determined.

[0104] Previous studies with mice have shown that both polar body genomes retain the potential to participate in normal development or form ESCs if reintroduced into the corresponding zygotic oocyte or cytoplasm (Wakayama S et al., Stem Cells 25, 986 to 993 (2007) and Wang T et al., Cell 157, 1591 to 1604 (2014), both of which are incorporated herein by reference). Here is described the

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23/55 translating these findings into mice and demonstrating that PBNT can rescue genetic material from human PB1 genomes discarded by development. Residual meiotic activity in enucleated Mil oocytes is sufficient to induce the formation of functional Mil-like spindles again that result in haploid oocyte after fertilization. Using non-invasive birefringence images, it was found that the majority of PBNT oocytes (67%) formed visible spindles within 60 min of fusion. However, a more detailed analysis of the spindle morphology in oocytes revealed that some meiotic spindles were in prophase or anaphase, suggesting compromised nuclear content in PB1. In addition, approximately half of the normally fertilized PBNT zygotes stop before reaching the blastocyst stage, suggesting that efforts to optimize protocols at the time of oocyte collection to isolate viable PBs are appropriate.

[0105] Aneuploidy in IVF embryos is relatively common (Alfarawati S et al., Fertil Steril 95, 520 to 524 (2011) and Angell RR et al., Nature 303, 336 to 338 (1983); incorporated herein by reference) and, as shown here, it also occurs in PBNT blastocysts. Aneuploidy rates in human embryos increase with maternal age as a result of meiosis I or II errors (Hassold T and Hunt P, Nat Rev Genet 2, 280 to 291 (2001); Nagaoka SI et al., Nat Rev Genet 13 , 493 to 504). (2012), Petronczki M et al., Cell 112, 423 to 440 (2003), incorporated by reference). In addition, mitotic errors can occur during post-zygotic divisions, producing mosaic blastomers containing multiple distinct karyotypes within a pre-implantation embryo (McCoy RC et al., PLoS Genetics 11, e1005601 (2015); incorporated by reference here). Although PBNT is unlikely to correct errors in meiosis I, the functional cytoplasm of young donors can reduce the incidence of aneuploidy resulting from meiosis II or mitotic errors. Aneuploidy rates in PBNT blastocysts normally fertilized in the present study were similar to controls based on STR and CGH-Arrangement analyzes. IS

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24/55 other optimizations of the PBNT protocols are likely to reduce aneuploidy rates. However, the implementation of pre-implantation genetic screening (PGS) is crucial before clinical applications of PBNT oocytes.

[0106] Due to the current tendency to delay procreation in the western world, age-related infertility with concomitant decreases in the ovarian reserve is common (Bellieni CV, World J Clin Pediatr 1, 34 to 36 (2012); incorporated here by reference) . It is described here that the generation of additional oocytes rescuing PB1 would increase the yield of patient-related blastocysts available for transfer from a single stimulation cycle. This implies that the use of PBNT can significantly improve ART results and pregnancy rates, particularly for women of advanced age with decreased ovarian reserve. However, this therapy is based on the patient's availability of PB1 and is therefore not applicable to women who cannot produce mature oocytes (Bilgin EM and Kovanci E, Curr Opin Obst Gynecol 27, 167 to 174 (2015); Grynberg M and others, Fértil Steril 105, 13 a 19 (2016), both of which are incorporated herein by reference). In addition, PBNT technology will be limited to countries, such as the United States, where in vitro fertilization programs can coordinate oocyte donation cycles with donor compensation. Polar body biopsy is an established technique in IVF and its removal of oocytes from the patient does not affect fertilization and subsequent embryo development (Cimadomo D et al., Biomed Res Int 2016, 7193075 (2016). Since PBNT counts with donor oocytes, this strategy allows the complementation of PB genomes of older women with cytoplasts from young donors.

[0107] The expanded use of PBNT, in addition to spindle transfer (ST), could provide an additional technique to support mitochondrial replacement therapy (MRT); a promising approach to prevent maternal mtDNA-based disease transmission (Tachibana M et al., Nature 493, 627 to 631 (2013) and Tachibana M and

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25/55 others, Nature 461,367 to 372 (2009); both are incorporated by reference). In mice, it has been shown that minimal mtDNA transport can be achieved in offspring generated by polar body transfer (Wang T et al., Cell 157, 1591 to 1604 (2014) and Yamada M et al., Cell Stem Cell 18, 749 a 754 (2016), both of which are incorporated by reference). Data are described here that suggest that, together with ST, PBNT could potentially increase the number of reconstructed MRT oocytes for families with mtDNA-based defects. Similar to other MRT technologies, PBNT will have to meet the safety and efficacy requirements of regulatory agencies before approving routine clinical applications (Wolf DP and others, Trends Mol Med 21.68 to 76 (2015); incorporated by reference here ). In addition, PBNT provides a tool to study basic mechanisms of developmental biology related to female meiosis and expands the understanding of genetic stability in oocytes.

EXAMPLES [0108] The following examples are for illustration only. In view of this description, those skilled in the art will recognize that variations of these examples and other examples of the described invention are possible without unnecessary experimentation.

[0109] Example 1 - Formation of New Mil Spindles from PB1 Genomes [0110] It was first determined whether residual meiotic activity in enucleated human Mil oocytes is sufficient to induce the formation of morphologically normal Mil-like spindles from the PB1 genome, carrying a genetic complement of Mil chromosomes (Hou Y et al., Cell 155, 1492 to 1506 (2013), incorporated by reference). For this purpose, fresh Mil oocytes donated by and recovered from 11 healthy volunteers (25 to 31 years old) were used after protocols for controlled ovarian stimulation (COS) and follicular aspirations

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26/55 transvaginal.

[0111] Using immunochemistry with antibodies against a and β tubulins and 4 ', 6diamidino-2-phenylindole (DAPI) for DNA staining, it has been determined that intact PB1s in oocytes Thousand mature humans do not contain detectable metaphase spindles, possibly due to a rapid decline in meiotic factors after abstraction (Figure 1B). The reconstructed PBNT oocytes were produced by fusing round intact PB1 with Mil cytoplasts (Figures 1A and 1C). Noninvasive imaging indicated that the majority of PBNT oocytes (67%) formed visible spindles within 60 minutes of fusion (Figure 1 D). Antibody staining for α and β tubulins and DAPI demonstrated that all PBNT oocytes (n = 5) contained fusochromosomal complexes. However, only 2 experimental oocytes formed metaphasic spindles similar to the intact Mil oocytes. The remaining PBNT spindles were in prophase (n = 2) or anaphase (n = 1) (Figure 1B) probably reflecting the compromised nuclear content.

[0112] Example 2 - Resumption and completion of meiosis II in PBNT oocytes after fertilization with sperm [0113] When the functionality of meiotic spindles in reconstructed PBNT oocytes was examined after fertilization, by intracytoplasmic sperm injection (ICSI), the completion of the meiosis II was evidenced by the abstraction of PB2 and the formation of pronuclei. The majority of PBNT oocytes survived ICSI (97%; 31/32) and formed visible pronuclei (81%; 25/31) at rates similar to the unhandled intact controls (Table 1). Most of the fertilized PBNT zygotes (76%; 19/25) contained 2 pronuclei (PN) and a second polar body (PB2) indicative of normal meiosis II completion (Figure 1E). The remaining zygotes contained irregular numbers of pronuclei (1 or 3) or were unable to extrude PB2 (Figure 1E; Table 1). These results were slightly inferior to those of the control (Table 1) indicating that the majority of PBNT oocytes formed functional spindles capable of

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27/55 recap of meiosis II after fertilization.

Table 1

Completion of Meiosis II in PBNT Oocytes and Mil Control Oocytes intact after fertilization Group No. of oocytes Survival after ICSI (%) Fertilization rate Completion of normal meiosis II Completion of abnormal meiosis II 3 PN (%) 1 PN (%) 2 PN / OPB (%) PBNT 32 31/32 (97 ± 1) 25/31 (81 ± 8) 19/25 (76 ± 10) 2/25 (8 ± 10) 2/25 (8 ± 5) 2/25 (8 ± 7) Control 21 20/21 (95 ± 6) 17/20 (85 ± 8) 16/17 (94 ± 4) 0/17 (0) 1/17 (6 ± 4) 0/17 (0)

[0114] Example 3 - Pre-implantation development and ploidy of blastocysts

PBNT [0115] To examine developmental competence, normally fertilized PBNT zygotes and controls were grown to the blastocyst stage. Similar to controls, 95% (18/19) of the PBNT zygotes cleaved, 74% (14/19) reached d8 cells and 68% (13/19) formed compact morula stage embryos (Table 2). In the control group, 75% (12/16) of the zygotes reached blastocysts, while only 42% (8/19) of the PBNT zygotes did it (Student's t test, p <0.05, Figure 1F, 1G and Table 2 ). As embryonic aneuploidy resulting from numerical chromosomal abnormalities leading to implantation failure or spontaneous loss of pregnancy is common, regardless of the morphology of the blastocyst, PBNT (n = 2) and control (n = 3) blastocysts were subjected to the analysis of short repetitions in tandem (STR). Examination of microsatellite markers mapped to 22 autosomal human loci and an X-linked locus revealed that two sampled PBNT blastocysts contained normal diploid chromosomes that contributed from both the oocyte and sperm. These results minimized the possibility of errors in the meiotic chromosomal segregation of the oocyte. Three additional PBNT blastocysts underwent a tropectoderma biopsy and underwent a comparative array genomic hybridization analysis (Arrangement-CGH) and determined that two were normal, but one blastocyst was aneuploid (loss of 17). This result was

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28/55 comparable to controls with a three-tested aneuploid blastocyst (loss of

21).

Table 2

Pre-implantation development of PBNT and control zygotes Group No. of oocytes with normal meiosis II completion 2 cells (%) 8 cells (%) Compact morula (%) Blastocyst (%) PBNT 19 18/19 (95 ± 2) 14/19 (74 ± 17) 13/19 (68 ± 18) 8/19 (42 ± 9) ^a Control 16 16/16 (100) 15/16 (96 ± 4) 14/16 (88 ± 5) 12/16 (75 ± 10) ^b

^a ' ^b Different superscripts within a column indicate significant differences (P <0.05).

[0116] Example 4 - Genetic integrity in ESCs derived from PBNT embryos [0117] In an effort to better define the potential clinical utility of reconstructed haploid PBNT oocytes, an ESC lineage was derived from five PBNT blastocysts. The lineage was designated PBNT1 (Figure 1F). Another four ESC strains were developed from nine control blastocysts. This included hESO-14, which was a genetically related brother of PBNT 1, derived from the same egg and sperm donors. The lower efficiency of ESC derivation (20%, 1/5) of PBNT embryos compared to controls (44%, 4/9) may indicate poor quality of PBNT blastocysts, but the total number was low for definitive conclusions. An ESC strain (PBNT2) was derived from two blastocysts developed from abnormal PBNT zygotes (3PN). The cytogenetic G band revealed that ο PBNT1 and three of the four control ESCs exhibited normal diploid karyotypes, with no evidence of detectable numerical or structural chromosomal abnormalities (Figure 2A). The remaining control hESO-15 was aneuploid with chromosome 21 monosomy (Figure 2A). In addition, the STR analysis of PBNT1 and hESO-14 confirmed their normal relationship between ploidy and siblings (Table 3). How

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29/55 expected, ο PBNT2 exhibited a triple karyotype 66-69, XXX (Figure 2A). DNA fingerprinting by STR corroborated the presence of three alleles for chromosomes 2, 6, 13, 18 and 22 and four alleles on chromosome 12 in this cell line (Table 3). Based on the allelic inheritance analysis, the triploid karyotype was caused by failure to complete meiosis II with retention of PB2 genetic material. This was consistent with the observation of 3 pronuclei and failure to segregate a second polar body after fertilization in these zygotes. In addition, sperm also contributed to a second allele for chromosome 12 tetraploidy. Both the PBNT1 and the hESO-14 control lines maintained the typical ESC morphology and formed teratoma tumors containing cells and tissues from all three layers. germinative (Figures 5A and 6B).

Table 3 [0118] PBNT1, PBNT2 and hESO-14 Paternity Analysis determined by Short Tandem Repeat Assay (STR)

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30/55

Loci STH, ÍW1 hgS044 Oocyte donor 1 4 sperm donor Oocyte donor 2 Sperm donor 2 Ssx f F : F 84 9 F 65 XX XX XX X ¥ XXX XX X ¥ 362 / Ϊ76 152/174 182 / Ϊ® iw.m iWi® 352/112 G2S1.33? ww 259/317 2"/"? »!« 23% w / w 233/203 3/3 52/163 392/2 « 2W369 192/192 iWm 146/369 113523.55 W3® M§S Wit » iwm WWÍ 2W3CS3 2S6 / ãS « MSttá 323/161 333/3 « 21/1 & 1 123/223 liWs 3.23 / 323 EXS14S7 123/123 3: 83/165 161/123 185/3 « 113/223 3.23 / 123 11W3 WW 227/2 « «7/1« W / 2S1 323/335 Í27 / Í3.S / 2S7 232/333 232/2 Si Ml » 172/112 WW IW / WÂ? 3 lSS / 172 3S4 / 322 013S2002 254/2 94 254/2 « 2W254 2W26Í 3W3S4 234/234 BIIS92S »& 88 392/3 « W & 88 39 // 299 262 / 36S 395/383 2S3 / 2S2 BS2S3M 3S4 / 2-SS 204/286 27S / 2-SS W2 / S 273/212/374 2/2/2 3W214 1 / 32S6.X 3W366 248 / 26S 248/264 253 / 2SS 256/304 Wl « 186/1 « W2 « 1WWW W206 366/152 : 4 // 543 133714s : 44/541 137/143 145/145 135 / W O1751380 201/265 285/165 W- / M1 26S / 2S9 332/2 » 257/265 EWS37 204/2 « 209/2 « tmsw 3.2S / 2 & S 2W2 « WS72 361/365 WS8? 363/3 » 38S / 362 361/395 WS » 301./38S D22S665 Ml® w » 164/169 1W1W1 « Wm iwm 03025/16 > 73/232 373/133 232/2 « 276 1W1 « Ww 162 ' MKY122 1W1CS W2 « 184/186 194/193 iWi » 1 «/ 132 3.06 / 163

Only male samples show a DXS2506 mark on the chromosome

X.

[0119] To assess whether PBNT introduced undetectable G-band subchromosomal changes, high resolution genomic physical maps across the genome of the PBNT1 and hESO-14 strains, as well as the egg donor and sperm donor DNA were generated. The BioNano Genomics Irys platform (Mak AC

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31/55 et al., Cell Stem Cell 5, 11 to 14 (2016); incorporated herein by reference) allows the detection of structural variations (SVs) including insertions, deletions and inversions in 1 kb resolution. When compared to the egg donor genome, 372 SVs were identified in PBNT1 and 380 SVs in hESO-14, of which 157 were shared between these sister cell lines (Figure 2B). In comparison with the sperm donor genome, 268 SVs were detected in PBNT1 and 265 SVs in hESO14 with 124 common SVs (Figure 2C). In addition, comparable numbers of unique SVs were identified in PBNT1 (281) and hESO-14 (297) (Figure 2D). No genes were found in or near SVs that were differentially expressed between PBNT1 and hESO-14.

[0120] Example 5 - Epigenetic and transcriptional signatures of PBNT ESCs [0121] DNA methylation involving the covalent transfer of a methyl group to the fifth carbon atom of the cytosine ring an important epigenetic mechanism that defines cell identity (Smith ZD and Meissner A, Nat Rev Genet 14, 204 to 220 (2013); incorporated here by reference. This process undergoes dynamic changes during and after fertilization, reflecting the reprogramming of development (Canovas S and Ross PJ, Theriogenology 86, 69 to 79 ( 2016); incorporated here as a reference.) In addition, the establishment of stable ESCs is associated with major changes in methylation profiles, as the genome reconfigures itself to the pluripotent state (Lister R et al., Nature 462, 315 to 322 (2009 ); Lister R et al., Nature 471, 68 to 73 (2011), both of which are incorporated herein by reference.) To assess the normality of these changes, DNA methylation analyzes were carried out at l ongo of the genome of the PBNT1 and hESO14 cell lines using high coverage base resolution MethylC-seq (Lister 2009 above). Both strains showed similar levels of CG methylation, a difference of only 0.7% between PBNT1 (86.1%) and hESO-14 (85.4%) and non-CG methylation, a difference of only 0.4% between PBNT1 (1.3%) and hESO-14 (0.9%) (Figure 3A and 3B).

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Pairwise comparisons of these two samples identified only 116 differentially methylated CG regions (DMRs) and 4 mega non-CG DMRs covering approximately 500kb (He Y and Ecker JR, Ann Rev Genom Hum Genet 16, 55 to 77 (2015); incorporated by reference here) (Figure 3C; Table S5, FDR <0.01). Visual inspection implied the formation of these DMRs (Figure 3D and 3E), but there was no enrichment in terms of genetic ontology (GO), genetic characteristics (Figure 3C) or correlation with the transcriptional profile. These results suggest that the overall methylation profile of the experimental PBNT1 was very similar to the control hESO-14.

[0122] Finally, global patterns of gene expression were examined in the PBNT1 and hESO-14 strains. Deep sequencing (50M readings) by RNAseq was performed in triplicates for each of the two strains. Differential expression analysis was performed using the edgeR package (Robinson MD and Smyth GK, Biostatistics 9, 321 to 332 (2008); incorporated here by reference) and showed that only 1.25% of the examined genes (186 out of 14,876) were differentially expressed between PBNT1 and hESO-14 (Figures 4A and 4B, FDR <0.05). Using the analysis of Genetic Ontology (GO), several GO terms were allocated. However, no statistically enriched functional categories were identified for the differentially expressed genes. These data indicated that the global patterns of gene expression of PBNT1 cells are similar to those of control hESO-14 cells.

[0123] Thus, within the limits of these analyzes, the PB1 genome interacts with the cytoplasm of the oocytes, establishing epigenetic and transcriptome characteristics that emulate the pluripotent state.

[0124] Example 6 - Gamete Donation [0125] Oocyte donors from 25 to 31 years of age were recruited through the OHSU Women’s Health Research Unit. Two healthy sperm donors

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33/55 were also recruited. Eligible participants participated in an information session describing the study's goals and related procedures. Written informed consent was obtained from all gamete donors stating that the oocytes will be fertilized to create embryos for research purposes and will not be used for reproductive purposes, but will be studied in vitro, including the generation of embryonic stem cells.

[0126] Example 7 - Methods [0127] To produce Mil receptor cytoplasty, oocytes were placed in a 50 μΙ manipulation droplet of human tubal fluid with 10% HEPES medium containing 5 pg / ml cytochalin B (HTF with HEPES 10% CB) in a glass bottom dish. The droplet was covered with tissue culture oil and the oocytes were maintained at 37 ° C for 10 min before enucleation. The dish was mounted on the stage of an inverted microscope (Olympus 1X71) equipped with a stage heater (http://www.tokaihit.com), Narishige micromanipulators, Oosight Imaging System (http://wwwcri-inc.com), and laser objective (http://www.hamiltonthome.com) (network sites and content incorporated here by reference). An oocyte was positioned using a holding pipette so that the spindle was in a position of approximately 2 to 4 hours. The pellucid zone near the spindle was laser drilled and then an enucleation pipette was introduced through the slit. The spindle was extracted by aspiration into the pipette with a minimal amount of cytoplasm and surrounding plasma membrane and discarded.

[0128] For the isolation of the polar body, an oocyte was positioned with the first polar body (PB1) at 2 am, and the pellucid zone was punctured before a transfer pipette was introduced and ο PB1 aspirated. Ο PB1 was then transferred briefly to a drop containing extract of HVJ-E (Ishihara Sangyo Kaisha) diluted 1: 3 with HTF with HEPES 10% CB, transferred and rinsed in HTF with HEPES 10% CB, and placed in the perivitelline space of the cytoplast receiver. The peers

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34/55 were washed in HTF with 10% HEPES, transferred to Global 10% medium, and incubated at 37 ° C in 6% CO2 for 60 minutes to allow fusion. The fusion was confirmed 30 to 60 min after nuclear transfer and the PBNT oocytes were fertilized after an additional 50 min culture using intracytoplasmic sperm injection (ICSI) using fresh donor sperm. The fertilized oocytes were then cultured in Global medium 10% 37 ° C in 6% CO2, 5% O2 and 89% N2. Fertilization was determined 16 hours after ICSI, observing the formation of pronuclei and the extrusion of the polar body. Un-manipulated Thousand Oocytes served as controls that were fertilized and cultured similarly to the PBNT group.

[0129] The following examples also help to demonstrate examples within the present scope regarding oocytes prepared to overcome problems with poor mitochondrial genomics.

[0130] Example 8 - Inclusion and Exclusion Criteria [0131] Since mitochondrial disease can be attributed to genome mutations in mtDNA and / or nuclear DNA (Wallace DC, J Clin Invest 123, 1405 to 1412 (2013); incorporated as a reference), an important clinical challenge is to confirm pathogenic mtDNA mutations in families eligible for MRT. Five families - four diagnosed with Leigh's syndrome (LS) and one with MELAS - were recruited and genetic tests carried out to confirm mtDNA mutations inherited from the mother. DNA was collected from blood, skin fibroblasts (SF) and / or urine from children and mothers, and complete mtDNA sequencing was performed. The first LS family had an affected 2-year-old child with a homoplastic T8993G replacement in both blood and SF samples, while his 22-year-old mother had the same mutation with 70% blood heteroplasm and 100% SF (Figure 6A , left).

[0132] In the second related LS family, an affected 2.5-year-old child carried the same T8993G mutation in 95% heteroplasmic blood and 100% in

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While in a second asymptomatic sibling aged 1 year, the mutation load was 50% in the blood and 62% in the SF. His 23-year-old mother, who was the older sister of the individual in the first family, carried this mutation by 13% in blood and 16% in SF (Figure 6A, right).

[0133] In the third LS family, the affected 12-year-old boy underwent a G13513A replacement in 56%, 86% and 97% of heteroplasmia in blood, SF and urine, respectively. Her asymptomatic 19-year-old brother had the same mutation in heteroplasma levels of 10%, 14% and 23% in blood, SF and urine, respectively. His 36-year-old mother also harbored the mutation at levels of 3%, 98% and 39% in blood, SF and urine, respectively (Figure 6B).

[0134] A fourth family also had a 1 year old child diagnosed with LS. However, genetic screening did not reveal any pathogenic mutations of mtDNA in the child or mother.

[0135] The fifth family comes from a large, well-studied strain of MELAS carrying an A3243G pathogenic mutation (Figure 10B) (McClelland K, 2014, https://scholarworks.csustan.edu/bitstream/handle/011235813/793/McClellandK. sum mer2014% 20.pdf? Sequence = 1; network site last accessed on November 28, 2016 and incorporated by reference in this document). The 32-year-old mother carried the mutation in 14%, 47% and 35% in blood, SF and urine, respectively. All of her 3 children inherited this mutation (Figure 6C).

[0136] These results indicated that pathogenic mtDNA mutations transmitted by the mother were implicated in four of the five families studied. The clinical LS phenotype in the fourth family was not associated with any pathogenic mtDNA mutation and, therefore, was excluded from further analysis. This highlights the importance of genetic testing for mtDNA diseases inherited by the mother before MRT. In addition, since levels of heteroplasm can vary between

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36/55 different tissues, it is crucial to sample and test blood, skin and urine, both in mothers and children (Monnot S et al., Hum Mutat 32, 116-125, 2011); incorporated here by reference).

[0137] Healthy voluntary oocyte donors were also screened and it was confirmed that they did not have any inherited pathogenic mtDNA mutations. Its mtDNA sequences and the corresponding haplotype were subsequently used for corresponding combinations of MRT.

[0138] Example 9 - COS (controlled ovarian stimulation), oocyte recovery and oocyte mutations [0139] Women with mtDNA disease exhibit rates of live births comparable to the general population, so it is accepted that they have normal fertility. However, the ovarian response to gonadotropin stimulation and oocyte recovery in women with pathogenic mtDNA mutations (carriers) was evaluated.

[0140] Age was similar between healthy carriers and donors. AMH levels, a measure of ovarian reserve, were lower in patients than in healthy donors (1.1 vs. 4.8 ng / ml). The count of antral follicles (AFC) was also lower in patients compared to healthy donors (10.3 vs. 22.3). In addition, the duration of COS was approximately one day longer in patients and the peak level of estradiol in the blood (E2) before the administration of hCG was also lower. Finally, the total number and number of mature Thousand oocytes recovered were also significantly lower in the carriers (5.8 vs. 16.6 and 3.8 vs. 13.2, respectively) (Figures 11 A, 11B, 11C, 11 D, 11E, 11F and 11G). It is noted that one carrier (cED2) exhibited premature luteinization, evidenced by the increase in progesterone levels before the LH outbreak (Figure 11H). Therefore, mutation analyzes were performed only on recovered atretic oocytes.

[0141] Although the numbers in the cohort were low (Smeets HJ and others,

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Ann NY Acad Sci 1350, 29-36 (2015); incorporated herein by reference), the results suggest that the mtDNA mutation could lead to a reduction in the ovarian response. Advanced age and higher BMI in patients could also have affected the result (Kelsey TW and others, PLoS One 6, e22024 (2011) and Kelsey TW and others, Mol Hum Reprod 18, 79 to 87 (2012); both are incorporated herein by reference). Another potential contributing factor is the long-term hormonal contraception these women were in before ovarian stimulation.

[0142] MtDNA mutations were then identified in individual carriers' oocytes. After removing the Mil spindle (nuclear genome), cytoplasts (mtDNA) were used for sequencing. In the first carrier, two retrieved oocytes contained the T8993G mutation at levels of 86% and 96%. In the second sibling, the levels of heteroplasm in 5 atresic oocytes ranged from 72% to 100%. In 4 oocytes from the third carrier of LS, the levels of G13513A mutation in 3 eggs were very low (0.6 to 3%), while in the fourth it was 40%. Finally, in nine oocytes of the MEL24 carrier A3243G, the mutation was not detected in an egg, while the rest contained 9 to 52% (Figure 7A). In the search for a method to predict mutation levels of oocytes, the mean levels of heteroplasmia in the blood of sister children were highly correlated with the levels of oocytes (Brown DT et al., Am J Hum Genet 68, 533 to 536 (2001) ; incorporated here by reference) (R ² = 0.52 vs. 0.85, Figure 7B).

[0143] Next, whole mtDNA sequences were analyzed in carrier oocytes in an effort to track secondary mtDNA mutations. Comparisons were made with oocytes from healthy donors. The carrier oocytes contained secondary heteroplastic variants of mtDNA, however, the mean number per oocyte was not significantly different from healthy controls (2.2 vs. 1.3; p> 0.05) (Figure 7C). Some of these variants were present in several oocytes from the same carrier and were also found in

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38/55 blood, skin or in sister children suggesting recurrent germline mutations. However, these variants were non-coding D-loop mutations. Others were found only in one oocyte within the group indicative of de novo mutations (45%). The majority (83%) of these secondary variants were low-level heteroplastic mutations (<15%) (Figure 111). The number of copies of MtDNA per oocyte was used as an indirect measure of oocyte quality (Monot S et al., Hum Mol Genet 22, 1867 to 1872 (2013) and Fragouli E et al., PLoS Genet 11, e1005241 (2015); incorporated here by reference). There were no significant differences between healthy and carrier oocytes (average of 348,954 vs. 283,605, respectively) (Figure 7D). A low number of mtDNA copies may play a role in reducing ovarian reserve (Boucret et al., Hum Reprod 30, 1653 to 1664 (2015); incorporated herein by reference), but no correlation between the number of oocyte copies and the AMH levels were observed here (Figure 11J).

[0144] In conclusion, the carrier oocytes were of normal quality and the levels of mutations in the blood of sister children appear to be of predictive value for the mutation in oocyte cohorts.

[0145] Example 10 - Spindle transfer (ST) in oocytes of carriers of mtDNA mutation [0146] Meiotic spindles (carioplasts; with maternal transport mtDNA) recovered from Mil carrier oocytes (n = 13) were transferred for enucleated donor oocytes (cytoplasts; donor mtDNA) (Figure 8A) while controls involved ST among healthy oocytes (n = 36) with pre-selected mtDNA haplotypes (Figure 12A). All karyoplasts and cytoplasts survived micromanipulation and fusion, with the exception of one in the control group. ST oocytes, together with intact (unhandled) controls, were then fertilized by intracytoplasmic sperm injection (ICSI) and cultured in blastocysts. High taxes

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39/55 of fertilization, comparable to intact controls, were observed in both ST groups (Figure 8A and Figures 12B and 12C). The subsequent development of ST diploid zygotes for the blastocyst stage was 75%, similar to controls. A similar pattern of development was observed among ST patients with different types of mutations (8993ST, 13513ST and 3243ST) (Figure 8B).

[0147] To address COS asynchrony, ideally, both donor carriers and healthy donors would undergo recovery on the same day with the end point of a similar number of mature oocytes for ST. An alternative approach to achieving these goals involves freezing, storing and thawing oocytes. ST between fresh and vitrified oocytes (frozen ST) was conducted. The general fertilization rate using vitrified oocytes was comparable to fresh ST, while the formation of normal diploid zygotes was lower (Figure 8A). There were no differences in fertilization between the vitrification of the carioplasty and the cytoplasty (Figure 12D).

[0148] Since the risk of communication failures between nuclear and mitochondrial genomes has led to concerns about potential secondary metabolic dysfunction (Woodson JD & Chory J, Nat Rev Genet 9, 383 to 395 (2008); incorporated herein by reference) , the development of the ST embryo was analyzed as a function of the donor mtDNA sequence distance. ST oocytes and embryos were grouped based on the number of single nucleotide polymorphisms (SNPs) between the original (maternal) mtDNA and the oocyte donor mtDNA, ranging from the next (6 SNPs) to the middle (33 SNPs) and the distant (57 SNPs) (Figure 12A). Fertilization and blastulation rates were similar between the three ST groups, suggesting that embryonic development is not compromised (Figure 8C). ESC lines were established from ST blastocysts and differentiated into neural progenitor cell cardiomyocytes (NPCs) and teratomas. Comparable differentiation efficiencies have been observed between the enzyme activity of

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40/55 mitochondrial respiratory chain (CR) and oxygen consumption rates (OCR) between ESC derivatives containing 6 or 49 SNP differences (Figures 8D, 13A, 13B, 16A and 16B). Interestingly, cells differentiated from an ST-ESC strain with 33 SNP differences showed significantly reduced OCR levels, alluding to possible nuclear to mitochondrial incompatibility.

[0149] In summary, MRT in oocytes carrying ST resulted in high fertilization and development of blastocysts similar to controls. In addition, the embryonic development, derivation and differentiation of ESC were not affected by the genetic background of the donor's mtDNA. However, it is possible that a given donor mtDNA may not function properly, a process that was independent of the total SNP differences.

[0150] Example 11 - Nuclear Genome Abnormalities [0151] Since ST embryos may be at risk for chromosomal or subchromosomal abnormalities, expanded blastocysts derived from ST and control embryos were biopsied and examined by CGH (comparative genome hybridization) . A slightly higher rate of aneuploidy was observed in both ST groups compared to intact controls, but the difference was not significant. This may be related to the higher maternal age in the group of patients (Figures 14A and 17). Then, a karyotype analysis of G bands in ST and control ESCs was performed. Abnormal karyotypes were observed in all groups, but no significant differences were found. G bands also detected cases of chromosomal mosaicism and polyploidy (Figures 14B and 18). Thus, to detect chromosomal abnormalities, the preimplantation genetic diagnosis can be performed before the transfer of the MRT embryo.

[0152] Copy number variations (CNVs) were examined in selected ST-ES cell lines to explore possible subchromosomal abnormalities (deletion or duplication) (Lupski JR, Trends Genet 14, 417 to 422

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41/55 (1998) and Sharp AJ et al., Nat Genet 38, 1038 to 1042 (2006), both of which are incorporated herein by reference). Again CNVs were detected in both controls, ST and intact, but were considered of uncertain clinical significance (Kearny HM et al., Genet Med 13, 680 to 685 (2011); incorporated herein by reference).

[0153] In summary, a total of 6 ST blastocysts were produced from 4 cycles of ovarian stimulation in patients (Table 4). One blastocyst was aneuploid and four were eligible for transfer based on morphological quality assessments. These results were comparable to controls.

[0154] Table 4. MRT results for families with pathogenic mtDNA mutations

Family 1 Family 2 Family 3 Family 4 Family 5 Mitochondrial disease LS LS LS LS MELAS Pathogenic mtDNA mutation T8993G T8993G G13513A Not A3243G Carrier age (years) 22 23 36 28 32 WAISTBAND Yes Yes Yes Deleted Yes No. of retrieved oocytes 3 5 4 at 11 No. of ST oocytes 2 Canceled 4 at 7 ST blastocysts 0 at 2 at 4 Grade at D6 at at 5AA 5BB at 5AA 5AA 5BB 5CC Aneuploidy at at Yes: +9 n / t at n / t Not Not Not Sex at at M at at at M F F MtDNA transport at at <1% at at at <1% <1% 1%*

Six ST embryos derived from oocytes carrying pathogenic mtDNA mutations have reached the blastocyst stage. At least 2 blastocysts were eligible for transfer. *, mtDNA in ESCs from that blastocyst reverted to a maternal mtDNA haplotype, but it was naturally occurring (did not carry A3243G mutation). n / a, not applicable; n / t, not tested.

[0155] Example 12 - Maintenance of donor mtDNA in ST embryos and

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42/55 ESC strains [0156] The efficacy and safety of MRT depend not only on the degree of maternal mtDNA initially co-transferred with the karyoplast, but also on its subsequent persistence and possible amplification in embryos. Recent studies have observed that, due to genetic drift, some MRT ESCs have restored maternal mtDNA (Yamada M et al., Cell Stem Cell 18, 749 to 754 (2016) and Hyslop LA et al., Nature 534, 383 to 386 (2016); which are hereby incorporated by reference). Clearly, a small amount of maternal mtDNA transport is common during TS resulting in low heteroplasm in human embryos and offspring of non-human primates (typically below 2%) (Pauli D et al., Nature 493, 632 to 637 (2013) ; incorporated here by reference). Here, all examined ST zygotes and cleaving embryos (n = 22) contained> 99% of donor mtDNA (Table 5) and similar results were observed in 13 of the 15 ES cell lines (87%) established from ST blastocysts controlled, regardless of the donor's mtDNA haplotype. However, 2 ESC strains of ST sibling embryos (ST-ES7 and ST-ES8), generated by the combination of maternal U5a and donor H1 b mtDNA (33 SNPs), showed high levels of maternal mtDNA (81% and 94%) (Table 5). The prolonged passage resulted in a complete loss of the donor's mtDNA and returned to the original maternal mtDNA haplotype (100%). Interestingly, two other genetically related ESC strains (ST-ES5 and ST-ES6) derived from the reciprocal combination of maternal H1 b and donor U5a showed high maintenance of donor mtDNA (> 99%). Another ESC strain generated from maternal U5a and donor mtDNA V3 (ST-ES9; 33 SNPs) also carried predominantly donor mtDNA.

[0157] Table 5. mtDNA from donor in pre-implantation human MRT embryos and ESC lines

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[0158] Among the three carrier ST-ESC strains, 3243ST-ES1 also contained a significant level of maternal mtDNA (20%) (Table 5). However, this maternal H49 haplotype did not include the A3243G mutation. The levels of maternal mtDNA in this cell line gradually increased during the extended culture to

90% in passage 8 and for homoplasm on p.10 (Figure 9A). Notably, another sibling ESC strain (3243ST-ES2) generated by the same combination of maternal and donor mtDNA did not show any increase in maternal mtDNA,

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44/55 presumably due to stochastic or bottleneck effects (Table 5).

[0159] The study was expanded to include eight SCNT-derived ESC strains (NT-ESCs) that also carry donor oocyte mtDNA (Table 5) (Tachibana M et al., Cell 153, 1228 to 1238 (2013); Ma H and others, Nature 524, 234-238 (2015) and Kang E and others, Cell Stem Cell 18, 625 ^to 636 (2016); incorporated herein by reference). The dynamics of mtDNA heteroplasm was analyzed during the extended passage and identified that the NT-ES8 showed a gradual increase in maternal (somatic) mtDNA of 19% in foot. 2 to 100% in p.10 (Table 5, Figure 15A). Note that this NT-ESC strain was generated by a combination of maternal mtDNA X2c and donor D4a haplotypes (39 SNPs).

[0160] In conclusion, the ST allows efficient replacement of the oocyte's mtDNA with very low transported mtDNA in the resulting pre-implantation embryos. Most ESCs derived from ST blastocysts maintained predominantly the donor's mtDNA. However, some ESC strains showed gradual loss of donor mtDNA and reversion to maternal mtDNA, requiring further research to better understand the underlying mechanisms.

[0161] Example 13 - Molecular and cellular mechanisms that contribute to the maintenance of donor mtDNA [0162] Based on the observation that combinations of specific haplotypes led to the reversion to maternal mtDNA, it was assumed that this could be due to replication preferential use of specific mtDNA haplotypes or that certain mtDNAs endowed cells with growth advantages (Burgstaller JP et al., Mol Hum Reprod 21, 11 to 22 (2015) and Agaronyan K et al., Science 347, 548 to 551 (2015), both incorporated herein by reference).

[0163] The highly polymorphic D-loop region of mtDNA, called conserved sequence II box (CSBII) was focused on first. It has previously been shown that a rare polymorphism in CSBII, that is, G5AG7, affects efficiency

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45/55 of mitochondrial transcription termination and generation of replication primers. When donor mtDNA and maternal mtDNA were compared in a total of 26 ESC strains with 18 different combinations of haplotypes, it was observed that two “reverse” brothers ST-ES7 and ST-ES8 carried donor mtDNA with a G5AG8 polymorphism while the maternal mtDNA was G6AG8. Using in vitro transcription assays, it was determined whether the synthesis of the replication primer by mitochondrial RNA polymerase was affected in the donor's mtDNA. It was found that the deletion of a single guanosine residue (G5AG8 vs. G6AG8) in the donor mtDNA results in a 4-fold reduction in the replication primer synthesis (Figure 9a). Therefore, certain mtDNA haplotypes varying in the CSBII sequence provide a more efficient synthesis of the replication primer and, subsequently, can achieve a replicative advantage. Without being limited by theory, this mechanism could explain the phenomenon of preferential replication of maternal mtDNA in some of the ESC strains. Sequence analysis found no differences in CSBII SNP in the remaining two 3243ST-ES1 and NT-ES8 reversed. Several other D-loop polymorphisms have been observed, including the TAS nucleus region, which is also involved in the regulation of mtDNA replication (Jemt E et al., Nucl Acids Res 43, 9262 to 9275 (2015); incorporated herein by reference) . Although the exact mechanism by which these polymorphisms affect replication remains unclear, it can be speculated that, similar to the CSBII and TAS regions, some D-loop variants may result in replication bias for a particular mtDNA haplotype.

[0164] It was also considered whether the additional independent replication mechanisms affect the reversion to maternal mtDNA and examined the increase in maternal mtDNA by 3243ST-ES1 and NT-ES8 reversed in whole cultures or individual cell clones. The initial levels of maternal mtDNA increased with the passage in whole cultures (Figures 9B, 9C and 15A). However, when ESCs were disassociated in individual cells and clones

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46/55 isolated from individual cultured cells, the initial levels of maternal mtDNA varied in different clones, but the heteroplasm did not change with the extended culture (Figure 9D). Significant differences in cell proliferation and growth rates between different clones were observed. Clones with higher levels of maternal mtDNA exhibited significantly faster growth rates than clones with lower maternal mtDNA heteroplasm (Figure 9E). These results suggest that certain mtDNA haplotypes give ESCs more rapid growth and proliferative advantages. Thus, in mixed cultures consisting of cells with varying heteroplasm, a subpopulation of cells with greater maternal mtDNA can overgrow cells with donor mtDNA.

[0165] The enzyme activities of complex I and complex IV of the mitochondrial respiratory chain were measured in cells carrying several mtDNA haplotypes, but no significant differences were found. This suggests that the reversion to maternal mtDNA is independent of mitochondrial activity (Figure 15B).

[0166] Next, it was examined whether the reversal is specific for non-differentiated ESCs or could occur during differentiation. Several reversed and non-reversed ESCs were differentiated in vitro into NPCs and cardiomyocytes, and in teratomas in vivo. Maternal mtDNA levels were measured before and after differentiation (Figures 16A, 16B). The levels of maternal mtDNA in reversed 3243ST-ES1 reduced to undetectable levels in some tissues, but were slightly elevated in other cell types compared to the initial 4% in undifferentiated ESCs (Figure 9F). Maternal mtDNA was undetectable during in vivo and in vitro differentiation of the 3243ST-ES2 non-reversed sibling cell line. In contrast, maternal mtDNA levels increased in all differentiated tissues tested in reversed ST-ES7 and NT-ES8 (Figure 9F). These results demonstrate that the mtDNA reversal phenomenon is not exclusive to ESCs and

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47/55 occurs during differentiation, increasing the possibility that the return to the mutant mtDNA may occur in some MRT children.

[0167] Example 14 - Conclusions [0168] Practicality and clinical results after MRT in oocytes carrying pathogenic mtDNA mutations have been investigated 8, 10. Since mutations in nuclear genes or mtDNA can cause similar mitochondrial syndromes, it is essential to perform genetic tests to confirm the eligibility of women with pathogenic DNA mutations for MRT.

[0169] A range of heteroplasm for the mutant mtDNA was observed between the different tissues tested on women carriers and their children. As expected, pathogenic mtDNA mutations were detected in oocytes from all carriers.

[0170] Maternal mtDNA transport, although detectable, was less than 1% in pre-implantation ST embryos. However, reversion to the original maternal mtDNA was observed in some MRT ESCs (15%, 4/26) during their extended culture or differentiation. Maternal mtDNA in ST monkeys was at expected low levels and did not increase with age in adults. However, a moderate increase in maternal mtDNA (16%) was observed in oocytes selected from ST females (Lee HS et al., Cell Rep 1.506 to 515 (2012); incorporated here by reference). This implies that, despite the efficient replacement of mutant mtDNA in oocytes, some MRT children may still develop high loads of mutation and mitochondrial disease.

[0171] Among the factors that may contribute to mtDNA reversal, it appears that this phenomenon was independent of MRT methods (ST, PNT or SCNT), presence of pathogenic mtDNA mutations and the genetic distance measured in total SNPs between donor mtDNA and maternal mtDNA. However, some combinations of specific haplotypes produced preferential increases in mtDNA

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48/55 maternal and gradual loss of donor mtDNA. One of the possible mechanisms responsible for the observed reversal could be the synthesis of more efficient replication primers and, thus, inclination towards the preferential amplification of mtDNA haplotypes with specific polymorphisms of the CSBII sequence. Selecting compatible donor mtDNA containing CSBII or other Dloop sequences similar to maternal mtDNA would solve this problem. Although the number of combinations tested in the study described is small, the possible order of replication advantage in the mtDNA haplotypes can be H56> H1b, U5a> H1b> F1a, U5a> X2c> D4a and H49> B2k. However, stochastic or bottleneck amplification of mtDNA during early embryonic development can also coexist.

[0172] Mitochondria not only generate energy, they also regulate cell proliferation and apoptosis (Chan DC et al., Cell 125, 1241 to 1252 (2006); incorporated herein by reference). It is described here that mtDNA haplotypes can affect cell growth and proliferation, thus providing selective advantage for cells with maternal mtDNA. Future applications of MRT will require additional studies evaluating mtDNA haplotypes from compatible donors to prevent OXPHOS dysfunction, differences in mtDNA replication, cell growth and recovery of mutant mtDNA (Koehler CM et al., Genetics 129, 247 to 255 (1991); incorporated here by reference).

[0173] Example 15 - mtDNA sequencing [0174] Specific mtDNA mutations and general haplotypes were determined in oocytes, blood, skin fibroblasts (SF) and / or urine by complete mtDNA sequencing using MiSeq or refractory mutation system amplification - quantitative polymerase chain reaction (ARMSqPCR).

[0175] Example 16 - Controlled Ovarian Stimulation (COS)

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49/55 [0176] Baseline screening was performed including a medical history and physical examination, assessment of body mass index (BMI), antimullerian hormone level (AMH) and antral follicle count (AFC). The individuals were submitted to ovarian stimulation and oocyte recovery, using standard IVF protocols and procedures.

[0177] Example 17 - Spindle Transfer (ST) [0178] The ST was performed as follows: meiotic metaphasic spindles II (Mil) were visualized under polarized microscopy and isolated with minimal cytoplasm (karyoplast, mtDNA host). The karyoplast was then placed in the perivitelline space of an enucleated oocyte (cytoplasm, donor mtDNA) and fused using HVJ-E (enveloped hemagglutinating virus from Japan).

[0179] Example 18 - Oocyte Vitrification [0180] Oocyte vitrification was performed as previously described using commercially available human oocyte vitrification and thawing kits (Vit. Kit and Vit. Heating kit, Life Global).

[0181] Example 19 - Derivation of ES Cells and Culture [0182] ST blastocysts were released from their pellucid zone and plated in mEF feed layers in 4-well culture plates for 5 to 7 days at 37 ° C, 3% CO2 , 5% O2 and 92% N2 in medium (DMEM / F12 with 10% FBS, 10% KSR, 0.1 mM non-essential amino acids, 1 mM l-glutamine, 0.1 mM β-mercaptoethanol, 5 ng / ml basic fibroblast growth factor, 10μΜ ROCK inhibitor). The attached blastocyst excrements were manually cut into small pieces, re-plated on new plates and kept in DMEM knockout medium (Invitrogen) supplemented with 20% KSR, 0.1 mM non-essential amino acids, 1 mM l-glutamine, β-mercaptoethanol at 0.1 mM, penicillin - streptomycin and 4 ng / ml bFGF for further propagation and analysis. All cell lines were negative for mycoplasma contamination.

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50/55 [0183] Example 20 - mtDNA transcription assays [0184] In vitro antitermination transcription assays were performed using PCR amplified models containing the donor (H1b) or host (U5a) mtDNA region 202-481. The transcription reaction products were resolved by 20% PAGE containing 6 M urea and visualized by PhosphoImager (GE Health).

[0185] Example 21 - Cell Growth and Proliferation Assays [0186] ESCs were adapted to grow under feeding-free conditions in the Matrigel matrix in mTeSRTMI medium (STEMCELL technologies) (Wu J et al., Nature 521,316 to 321 (2015) ; incorporated herein by reference). The cells were dissociated with Accutase (Life Technologies) for 2 min and approximately 10 ⁵ cells were seeded on each 60 mm plate. The cells were harvested periodically and counted using the Automatic Condensed Cell Counter (Invitrogen).

[0187] Example 22 - Differentiation and Culture of Neural Progenitor Cells (NPC) [0188] A previously published protocol with minor modifications was used for the differentiation of NPC. Briefly, ESCs were maintained in MEFs in CDF12 medium prior to NPC differentiation. The CDF12 medium contained DMEM / F12 (Life Technology), 20% knockout serum replacement (Life Technologies), 2 mM Glutamax (Life Technologies), 0.1 mM NEAA (Life Technology), 0.1 mM β -mercaptoethanol (Life Technologies) and 4 ng / ml FGF2 (Peprotech). ESCs were disaggregated with Collagenase IV (Life Technologies) and allowed to grow to approximately 40% confluence. To initiate neural induction, cells were washed twice with 1x DPBS without calcium and magnesium (Corning Cellgro) and changed to Neural Induction Medium 1 (NIM-1: 50% Advanced DMEM / F12 (Invitrogen), 50% Neurobasal ( Invitrogen), 1x B27 (Invitrogen), 1x N2

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51/55 (Invitrogen), 2 mM GlutaMAX and 10 ng / ml hLIF (Peprotech), 4 µM CHIR99021 (Selleckchem), 3 µM SB431542 (Selleckchem), 2 µM Dorsomorphine (Sigma) and 0.1 µM Compound E (SEM Chemicals Inc.). After 2 days of culture in the NIM-1 medium, the cells were switched to Neural Induction Medium 2 (NIM-2: 50% Advanced DMEM / F12, 50% Neurobasal, 1x N2, 1x B27, 2mM GlutaMAX and 10 ng / ml hLIF, 4 µM CHIR99021.3 µM SB431542 and 0.1 µM Compound E.) The cells were further cultured in NIM-2 for 5 days with a change of medium daily. On the last day in the NIM-2 medium, the cells were treated overnight with 10 µM Y27632 (Selleckchem) and 20 to 30 “dome” colonies were harvested manually and digested with Accumax (Innovative Cell Technologies) for 10 minutes at 37 ° C. Accumax treatment cells were gently disaggregated into individual cells and plated again in 6-well plates coated with Matrigel at a density of approximately 3.5 x 10 ⁴ per cm ² in Cell Maintenance Medium Neural progenitors (NPMM: 50% Advanced DMEM / F12, 50% Neurobasal, 1x B27, 1x N2, 2 mM GlutaMAX, 10ng / ml hLIF, 3 mM CHIR99021 and 2 mM SB431542) supplemented with 10 uM Y27632. The NPCs were maintained on plates coated with Matrigel in NPMM. The NPCs were passed when they reached approximately 70% to 80% confluence using Accumax and re-plated at a density of 3 x 10 ⁴ per cm ² with average daily change. For the initial 6 passages, the NPCs were pretreated with Y27632 at 10 μΜ during the night before and during cell division. The study was randomized and the researchers were blinded to treatment allocations in collaborators' laboratories.

[0189] Example 23 - Cardiomyocyte Differentiation [0190] Cardiomyocyte differentiation was performed based on a GiWi protocol (GSK3 inhibitor and Wnt inhibitor) described previously (Lian X et al., Nat Protoc 7, 1235 to 1246 (2012 ); incorporated herein by reference).

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Briefly, ESCs were cultured in Matrigel-coated plates in mTeSRI medium at 80-90% confluence prior to passage with Accutase and then seeded again in 0.5-1.5 million cells per well in 12-well plates in 1 ml of mTeSRI plus 10 μΜ of Y27632. On day 5, the cells were incubated with 6 to 12 μΜ of CHIR99021 (Selleckchem) for 16-24 h and then replaced with 2 ml of RPMI / B27 (insulin) and cultured for two days. On day 8, a 2 ml combined medium was prepared by mixing 1 ml of medium collected from the 12-well plate and 1 ml of fresh RPMI / B27 (-insulin) medium. The cells were then replaced with 2 ml of medium combined with 5 μΜ of IWP2 (Tocris) and cultured for 48 h. On day 10, 2 ml of fresh RPMI / B27 (insulin) was added to each well of the 12-well plate. From day 12, the medium was changed every three days for 2 ml / well of RMPI / B27 (+ insulin). The contracted cardiomyocytes were already observed on the 17th from the initial passage of ESCs. Differentiated cell types were identified by immunocytochemistry as previously described.

[0191] Example 24 - Teratoma Assay [0192] ESCs were injected into the femoral region of 6 week old male SCID mice (Charles River). Mice with tumors were euthanized and teratomas were isolated, sectioned and histologically characterized for the presence of representative tissues as previously described.

[0193] Example 25 - Assessment of Mitochondrial Function [0194] The enzymatic activities of the mitochondrial respiratory chain (complexes I and IV) were measured by spectrophotometry, as described, for example, by Spinazzi M et al., Nat Protoc 7, 1235 to 1246 (2012); incorporated here by reference. Briefly, intact mitochondria were isolated from fibroblasts and treated with and without 3 mM rotenone at a wavelength of 340 nm for 5 min. Differences in absorbance per minute were obtained, and the specificity of

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53/55 COMI activity was estimated by the percentage of rotenone inhibition. For oxygen consumption of living cells, an XF96 extracellular flow analyzer (Seahorse Biosciences) was used to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), as previously described. Briefly, neural progenitor cells (NPCs) were seeded at a density of 30,000 cells per well of an XF96 cell culture microplate and incubated for 24 h to allow the cells to bind. Prior to the trial, NPCs were equilibrated for 1 hr in non-buffered XF assay medium supplemented with 25 mM glucose, 1 mM sodium pyruvate, 2 mM glutamax, 1x non-essential amino acids and 1% (by volume) FBS in a non-C02 incubator. Sequential injections of oligomycin compound (0.5 μΙ / ml), carbonyl cyanide 4 (trifluoromethoxy) phenylhydrazone (FCCP, 1 μΜ) and rotenone (0.5 μΜ) / antimycin A (1 μΜ) were used to measure key parameters of mitochondrial breathing. mitochondrial function indices were calculated as baseline respiratory rate (baseline OCR of rotenone / antimycin A), dependent on ATP (basal respiratory rate - OCAR oligomycin), maximum respiratory rate (FCCP OCR - OCR rotenone / antimycin A) and oxidative reserve ( maximum breath rate - basal breath rate). Each value represented was the average of a minimum of 4 replicated wells, and was normalized to the total number of cells plated. The results were presented as mean ± SEM. Unidirectional ANOVA was used for three comparisons between groups and Student's t test was used for comparisons between pairs. A P value less than 0.05 was considered significant. The study was randomized, and the researchers were blinded to allocating samples between different groups.

[0195] Example 26 - Sequencing of mtDNA by MiSeq [0196] DNA was extracted from skin fibroblasts, whole blood, urine, ESCs and teratoma tissues using the Centra DNA extraction kit (Qiagen), and oocytes using the DNA extraction kit Pico Pure (Life Technologies). MtDNA was

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54/55 amplified by a single PCR reaction. The amplification of individual oocyte mtDNA was performed using 2 sets of primers: 7272F 5GGCTCATTCATTTCTCTAACAG-3, 15712R 5-TTGGCTTAGTGGGCGAAATA-3 and 15635F

[0197] Example 27 - Detection of mtDNA variants by Sanger sequencing [0198] The mtDNA variants present in more than 10% heteroplasm were independently corroborated by Sanger sequencing. MtDNA regions containing germline mutations or SNPs were amplified by PCR with sets of primers previously described using the PCR Platinum SuperMix high-fidelity kit (Life Technologies). The PCR products were purified, sequenced and analyzed by Sequencher v. 5.0 (GeneCodes).

[0199] Example 28 - Measurement of ARMS-qPCR and Number of Copies [0200] The quantitative PCR assay of the refractory amplification mutation system (ARMs-qPCR) was performed to check for heteroplasm in mt8993T> G, mt13513G> A and mt3243A> G e to measure transport in control ST embryos and ES cells in 7843A> G, 16519T> C and 16278T> C8. Measurement of the number of copies of mtDNA was performed.

[0201] Example 29 - Genetic and Cytogenetic Analysis [0202] G-band karyotyping was performed on 20 metaphasic cells from each human cell line. CGH arrangement was performed by IVI Gen. CNVs were identified by SNP genotyping as previously described by MaH et al., Nature 511, 177 to 183 (2014) incorporated by reference and analyzed by a clinical genetic diagnostic laboratory.

[0203] Example 30 - Statistical Analysis [0204] The results are presented as means ± SD, or SEM. p <0.05

Petition 870190064911, of 10/07/2019, p. 61/95

55/55 was considered significant. Data were analyzed using Pearson's nonparametric test for correlation, independent group t test or chi-square test for paired comparison and ANOVA with Bonferroni analysis for multiple comparisons (IBM SPSS).

Claims (17)

1. Method for preparing a viable human oocyte for fertilization, CHARACTERIZED by the fact that it comprises the steps of: a) isolate a first oocyte containing a polar body; b) gain access through the pellucid zone of the first oocyte; c) removing the polar body from the first oocyte; d) removing the nuclear spindle from a second oocyte to create an enucleated cytoplasm; e) placing the polar body removed from the first oocyte in the perivitelline space of the enucleated cytoplasm. 2. Method, according to claim 1, further characterized by the fact that it comprises fertilizing the viable human oocyte through in vitro fertilization, thus creating an embryo. 3. Method, according to claim 2, further characterized by the fact that it comprises implanting the embryo in a receptive uterus. Method according to any one of claims 1 to 3, CHARACTERIZED by the fact that the enucleation of the first oocyte is carried out in a medium comprising at least 10% of human tubal fluid, HEPES buffer, and cytochalasin B using a Polarization Microscope Imaging. Method according to any one of claims 1, 2 or 3, CHARACTERIZED by the fact that the isolation of the polar body is carried out in a medium comprising at least 10% of human tubal fluid, HEPES buffer, cytochalasin B, and fusion with donor cytoplasm induced with HVJ-E extract. 6. Method for producing a human oocyte in vitro, CHARACTERIZED by the fact that it comprises: a) determine a mitochondrial DNA sequence from sequence box II Petition 870190043447, of 05/08/2019, p. 7/25 2/3 preserved of a donor oocyte; b) enuclear the donor's oocyte while the donor's oocyte is trapped in metaphase II, thus isolating the donor's nuclear genetic material; c) introducing the donor's nuclear genetic material into an enucleated recipient's oocyte, provided that the donor's oocyte's mitochondrial DNA has the same conserved sequence II genotype as the recipient's oocyte. 7. Method, according to claim 6, further characterized by the fact that it comprises fertilizing the viable human oocyte through in vitro fertilization, thus creating an embryo. 8. Method, according to claim 7, further characterized by the fact that it comprises implanting the embryo in a receptive uterus. 9. Method according to any of claims 6, 7 or 8, CHARACTERIZED by the fact that the donor oocyte enucleation is carried out in a medium comprising at least 10% of human tubal fluid, HEPES buffer, and cytochalasin B using a Polarization Microscope Imaging System. 10. Method according to any of claims 2, 3, 7 and 8, CHARACTERIZED by the fact that the embryo is cultured in the blastocyst stage before implantation in a receptive uterus. 11. Method according to any of claims 6, 7, 8, 9 or 10, CHARACTERIZED by the fact that the donor oocyte and the recipient oocyte both have the G5A8 genotype or both have the G6A8 genotype. 12. Method according to any one of claims 6, 7, 8, 9 or 10, CHARACTERIZED by the fact that the donor oocyte's mitochondrial DNA comprises a mutation that results in a mitochondrial disease. 13. Method, according to claim 12, CHARACTERIZED by the fact that mitochondrial disease is selected from Leigh's Syndrome, Petition 870190043447, of 05/08/2019, p. 8/25 3/3 Mitochondrial encephalomyopathy, Lactic Acidosis or Stroke type episodes. 14. Method of producing a human oocyte in vitro, the human oocyte produced comprising minimal maternal mitochondrial DNA, CHARACTERIZED by the fact that it comprises: determining the sequence of the main non-coding region of the mitochondrial DNA of a donor oocyte, wherein the sequence of the main non-coding region includes a D loop region; enuclear the donor's oocyte while the donor's oocyte is trapped in metaphase II, thus isolating the donor's nuclear genetic material; Introduce the donor's nuclear genetic material into an enucleated recipient's oocyte, provided that the donor's oocyte's mitochondrial DNA has the same sequence as the main non-coding region of the recipient's oocyte. 15. Method, according to claim 14, CHARACTERIZED by the fact that the donor oocyte and the recipient oocyte both have the mtDNA H56, H1b, U5a, F1a, X2c, D4a, H49 or B2k haplotype. 16. Method, according to claim 14, CHARACTERIZED by the fact that the donor oocyte's mitochondrial DNA comprises a mutation that results in a mitochondrial disease. 17. Method, according to claim 16, CHARACTERIZED by the fact that mitochondrial disease is Leigh's syndrome, mitochondrial encephalomyopathy, lactic acidosis, or stroke-like episodes.